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J Allergy Clin Immunol ; 154(1): 195-208.e8, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38479630

RESUMO

BACKGROUND: X-linked agammaglobulinemia (XLA) is an inborn error of immunity that renders boys susceptible to life-threatening infections due to loss of mature B cells and circulating immunoglobulins. It is caused by defects in the gene encoding the Bruton tyrosine kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. This paper reports on a gene editing protocol to achieve "knock-in" of a therapeutic BTK cassette in hematopoietic stem and progenitor cells (HSPCs) as a treatment for XLA. METHODS: To rescue BTK expression, this study employed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system that creates a DNA double-strand break in an early exon of the BTK locus and an adeno-associated virus 6 virus that carries the donor template for homology-directed repair. The investigators evaluated the efficacy of the gene editing approach in HSPCs from patients with XLA that were cultured in vitro under B-cell differentiation conditions or that were transplanted in immunodeficient mice to study B-cell output in vivo. RESULTS: A (feeder-free) B-cell differentiation protocol was successfully applied to blood-mobilized HSPCs to reproduce in vitro the defects in B-cell maturation observed in patients with XLA. Using this system, the investigators could show the rescue of B-cell maturation by gene editing. Transplantation of edited XLA HSPCs into immunodeficient mice led to restoration of the human B-cell lineage compartment in the bone marrow and immunoglobulin production in the periphery. CONCLUSIONS: Gene editing efficiencies above 30% could be consistently achieved in human HSPCs. Given the potential selective advantage of corrected cells, as suggested by skewed X-linked inactivation in carrier females and by competitive repopulating experiments in mouse models, this work demonstrates the potential of this strategy as a future definitive therapy for XLA.


Assuntos
Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia , Linfócitos B , Edição de Genes , Doenças Genéticas Ligadas ao Cromossomo X , Células-Tronco Hematopoéticas , Agamaglobulinemia/genética , Agamaglobulinemia/terapia , Agamaglobulinemia/imunologia , Animais , Tirosina Quinase da Agamaglobulinemia/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Humanos , Linfócitos B/imunologia , Camundongos , Masculino , Transplante de Células-Tronco Hematopoéticas , Diferenciação Celular/genética , Sistemas CRISPR-Cas
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